ABSTRACT Regeneration of the thymus is a critical process that allows for renewal of immune competence following infection, shock, cytoreductive chemo- or radiation therapy and other causes of thymic injury. This is particularly relevant for recipients of allogeneic hematopoietic cell transplantation (allo-HCT), who experience prolonged post-transplant T cell deficiency caused by cytoreductive conditioning and graft-versus-host disease (GVHD), which results in increased morbidity and mortality from infections and malignant relapse. One of the major goals of this project is to identify novel pathways of endogenous thymic regeneration so that they may be exploited into clinically relevant strategies for immune rejuvenation. We have uncovered a novel role for thymic endothelial cells (ECs) in mediating thymic regeneration after insults. We found that following thymic insults radioresistant ECs increase their production of BMP4, which acts on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance and regeneration. These effects promote TEC regeneration and overall thymic reconstitution after injury. Accordingly, abrogation of BMP4 effects by either pharmacologic or genetic inhibition impairs thymic repair. Importantly, we demonstrated that the adoptive transfer of ex vivo expanded thymic ECs (exECThy) represents a feasible approach to deliver BMP4 in the thymus and enhances thymic regeneration after immune damage. In addition, we also found that oxidative stress byproducts, such as lipid peroxidation products, accumulate in the thymus after injury and instruct pro-survival signals to the ECs through their activation of the damage sensing receptor TRPA1. We demonstrated that TRPA1 agonists can enhance thymic regeneration through activation of ECs. Based on these findings, we hypothesize that (a) ECs play an important role in T cell regeneration following thymic insult, (b) pathways that promote EC survival and function can be employed to enhance thymic regeneration and (c) administration of exECs, BMP4 and/or TRPA1 agonists can be used as therapeutic strategies to enhance post-transplant immune reconstitution. We propose in Aim 1 to study the effects of exECThy and BMP4 administration to HCT recipients on thymic regeneration, peripheral T cell reconstitution, GVHD and graft-versus-tumor activity. In Aim 2 we will study the mechanisms involved in the damage-resistance of thymic ECs with a focus on a) lipid peroxidation products and TRPA1, and b) anti- oxidant pathways such as Nrf2. These mechanistic and pre-clinical studies have the potential to define important novel pathways in thymic regeneration, which could result in clinical approaches to enhance T cell immunity, not only for recipients of allo-HCT, but also for individuals with T cell deficiencies due to aging (lymphoid atrophy), autoimmune diseases, infectious diseases, shock, radio- or chemo-therapy and radiation injury.